The Journal of Membrane Biology

, Volume 210, Issue 3, pp 173–181 | Cite as

Mimetic Membrane System to Carry Multiple Antigenic Proteins from Leishmania amazonensis

  • Fabiana R. Santos
  • Denise B. Ferraz
  • Katia R. P. Daghastanli
  • F. Juarez Ramalho-Pinto
  • Pietro Ciancaglini


Liposomes have long been used as models for lipid membranes and for the reconstitution of a single or multiple proteins. Also, liposomes have adjuvant activity in vaccines against several protozoan or bacterial organisms. Thus, the main objective of the present study was to obtain a crude extract of detergent-solubilized proteins of Leishmania amazonensis amastigotes and reconstitute them into liposomes. Neutral and zwiterionic detergents were less efficient than an ionic detergent. In order to obtain efficient solubilization using only sodium dodecyl sulfate (SDS), the effects of detergent and protein concentration and incubation time were studied. The maximum of solubilized proteins was obtained instantaneously using a ratio of 0.5 mg/ml of protein to 0.1% (w/v) detergent at 4°C. Dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS) and cholesterol in a weight ratio of 5:1:4 were used for protein reconstitution into liposomes using the cosolubilization method, yielding 60% of incorporation. The incorporation of multiple parasite proteins results in a vesicular diameter of proteoliposomes of about 140 nm, presenting a final lipid weight ratio for DPPC, DPPS and cholesterol of 1:1:5, with high stability. The detergent-solubilized proteins of L. amazonensis amastigotes present in the proteoliposome, when analyzed by SDS-polyacrylamide gel electrophoresis, include a wide range of parasite-incorporated proteins. BALB/c mice inoculated with these proteoliposomes were able to produce antibodies against the proteins reconstituted in DPPC:DPPS:cholesterol liposomes and were partially resistant to infection with L. amazonensis promastigotes. These results indicate that this system can be used as a possible vaccine against L. amazonensis.


Leishmania amazonensis Membrane protein Detergent solubilization Liposome Proteoliposome Cosolubilization method 



The authors thank Ms. Priscila Cerviglieri for revision of the text manuscript. F. J. R. P. is an emeritus researcher of CNPq. We also thank FAPESP, CNPq and CAPES for the financial support given. F. R. S. and K. R. P. D. are recipients of a studentship from CAPES and FAPESP, respectively.


  1. Aebischer T., Wolfram M., Patzer S.I., Ilg T., Wiese M., Overath P. 2000. Subunit vaccination of mice against new world cutaneous leishmaniasis: comparison of three proteins expressed in amastigotes and six adjuvants. Infect. Immun. 68:1328–1336PubMedCrossRefGoogle Scholar
  2. Afrin F., Ali N. 1997. Adjuvanticity and protective immunity elicited by Leishmania donovani antigens encapsulated in positively charged liposomes. Infect. Immun. 65:2371–2377PubMedGoogle Scholar
  3. Afrin F., Anam K., Ali N. 2000. Induction of partial protection against Leishmania donovani by promastigote antigens in negatively charged liposomes. J. Parasitol. 86:730–735PubMedGoogle Scholar
  4. Afrin F., Rajesh R., Anam K., Gopinath M., Pal S., Ali N. 2002. Characterization of Leishmania donovani antigens encapsulated in liposomes that induce protective immunity in BALB/c mice. Infect. Immun. 70:6697–6706PubMedCrossRefGoogle Scholar
  5. Ali N., Afrin F. 1997. Protection of mice against visceral leishmaniasis by immunization with promastigote antigen incorporated in lipossomes. J. Parasitol. 83:70–75PubMedCrossRefGoogle Scholar
  6. Camolezi F.L., Daghastanli K.R.P., Magalhães P.P., Pizauro J.M., Ciancaglini P. 2002. Construction of an alkaline phosphatase-liposome system: a tool for biomineralization study. Int. J. Biochem. Cell Biol. 34:1091–1101PubMedCrossRefGoogle Scholar
  7. Cevc, G., March, D. 1987. Bilayer elasticity. In: E. E. Bittar editor. Phospholipid Bilayers, Physical Principles and Models. Cell Biology: A Series of Monographs, pp 347–368. Wiley, New York.Google Scholar
  8. Chen P.S., Jr., Toribara T.Y., Warner H. 1956. Microdetermination of phosphorus. Anal. Chem. 28:1756–1758CrossRefGoogle Scholar
  9. Courret N., Frehel C., Prina E., Lang T., Antoine J.C.. 2001. Kinetics of the intracellular differentiation of Leishmania amazonensis and internalization of host MHC molecules by the intermediate parasite stages. Parasitology 122:263–279PubMedCrossRefGoogle Scholar
  10. Daghastanli K.R.P., Ferreira R.B., Thedei G., Jr., Maggio B., Ciancaglini P. 2004. Lipid composition-dependent incorporation of multiple membrane proteins into liposomes. Colloids Surf B Biointerfaces 36:127–137PubMedCrossRefGoogle Scholar
  11. de Jonge M.I., Hamstra H.J., Jiskoot W., Roholl P., Williams N.A., Dankert J., van Alphen L., van der Ley P. 2004. Intranasal immunisation of mice with liposomes containing recombinant meningococcal OpaB and OpaJ proteins. Vaccine 22:4021–4028PubMedCrossRefGoogle Scholar
  12. Descoteaux A., Turco S.J. 1999. Glycoconjugates in Leishmania infectivity. Biochim. Biophys. Acta 1455:341–352PubMedGoogle Scholar
  13. Foged C., Arigita C., Sundblad A., Jiskoot W., Storm G., Frokjaer S. 2004. Interaction of dendritic cells with antigen-containing liposomes: effect of bilayer composition. Vaccine 22:1903–1913PubMedCrossRefGoogle Scholar
  14. Furth A.J., Bolton H., Potter J., Priddle J.D. 1984. Separating detergent from proteins. Methods Enzymol. 104:318–328PubMedCrossRefGoogle Scholar
  15. Greaves D.R., Gordon S. 2005. Thematic review series. The immune system and atherogenesis: recent insights into the biology of macrophage scavenger receptors. J. Lipid Res. 46:11–20PubMedCrossRefGoogle Scholar
  16. Gregoriadis G. 1990. Immunological adjuvants: a role for liposomes. Immunol. Today 11:89–97PubMedCrossRefGoogle Scholar
  17. Haaker H., Racker E. 1979. Purification and reconstitution of the Ca2+-ATPase from plasma membrane of pig erythrocytes. J. Biol. Chem. 254:6598–6602PubMedGoogle Scholar
  18. Harshyne L.A., Zimmer M.I., Watkins S.C., Barrat-Boyes S.M. 2003. A role for class A scavenger receptor in dendritic cell nibbling from live cells. J. Immunol. 170:2302–2309PubMedGoogle Scholar
  19. Hartree E.F. 1972. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal. Biochem. 48:422–427PubMedCrossRefGoogle Scholar
  20. Hespanhol R.C., de Nazaré C., Soeiro M., Meuser M.B., de Nazareth S.L., Meirelles M., Corte-Real S. 2005. The expression of mannose receptors in skin fibroblast and their involvement in Leishmania (L.) amazonensis invasion. J. Histochem. Cytochem. 53:35–44PubMedCrossRefGoogle Scholar
  21. Higgins, G. 1987. Separation and analysis of membrane lipid components. In: J. B. C. Findlay, W. H. Evans, editors. Biological Membranes. Pratical Approach Series, pp 104–137. IRL Press, OxfordGoogle Scholar
  22. Ilgoutz S.C., McConville M.J. 2001. Function and assembly of the Leishmania surface coat. Int. J. Parasitol. 31:899–908PubMedCrossRefGoogle Scholar
  23. Jones O.T., Eubanks J.H., Earnest J.P., McNamee M.G. 1988. Reconstitution of the nicotinic acetylcholine receptor using a lipid substitution technique. Biochim. Biophys. Acta 944:359–366PubMedCrossRefGoogle Scholar
  24. Kita Y., Tanaka T., Yoshida S., Ohara N., Kaneda Y., Kuwayama S., Muraki Y., Kanamaru N., Hashimoto S., Takai H., Okada C., Fukunaga Y., Sakaguchi Y., Furukawa I., Yamada K., Inoue Y., Takemoto Y., Naito M., Yamada T., Matsumoto M., McMurray D.N., Cruz E.C., Tan E.V., Abalos R.M., Burgos J.A., Gelber R., Skeiky Y., Reed S., Sakatani M., Okada M. 2005. Novel recombinant BCG and DNA-vaccination against tuberculosis in a cynomolgus monkey model. Vaccine 23:2132–2135PubMedCrossRefGoogle Scholar
  25. Laemmli U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  26. le Maire M., Champeil P., Moller J.V. 2000. Interaction of membrane proteins and lipids with solubilizing detergents. Biochim. Biophys. Acta 1508:86–111PubMedCrossRefGoogle Scholar
  27. Lezama-Davila C.M. 1997. Vaccination of different strains of mice against cutaneous leishmaniosis: usefulness of membrane antigens encapsulated into liposomes by intraperitoneal and subcutaneous administration. Arch. Med. Res. 28:47–53PubMedGoogle Scholar
  28. Mazumdar T., Anam K., Ali N. 2004. A mixed Th1/Th2 response elicited by a liposomal formulation of Leishmania vaccine instructs Th1 responses and resistance to Leishmania donovani in susceptible BALB/c mice. Vaccine 22:1162–1171PubMedCrossRefGoogle Scholar
  29. Mitchell G.H., Thomas A.W., Margos G., Dluzewski A.R., Bannister L.H. 2004. Apical membrane antigen 1, a major malaria vaccine candidate, mediates the close attachment of invasive merozoites to host red blood cells. Infect. Immun. 72:154–158PubMedCrossRefGoogle Scholar
  30. Noronha F.S., Nunes A.C., Souza K.T., Melo M.N., Ramalho-Pinto F.J. 1998. Differential sensitivity of new world Leishmania spp. promastigotes to complement-mediated lysis: correlation with the expression of three parasite polypeptides. Acta Trop. 69:17–29PubMedCrossRefGoogle Scholar
  31. Nunes A.C., Almeida-Campos F.R., Horta M.F., Ramalho-Pinto F.J. 1997. Leishmania amazonensis promastigotes evade complement killing by interfering with the steps of the cascade. Parasitology 115:601–609PubMedCrossRefGoogle Scholar
  32. Parmar M.M., Edwards K., Madden T.D. 1999. Incorporation of bacterial membrane proteins into liposomes: factors influencing protein reconstitution. Biochim. Biophys. Acta 1421:77–90PubMedCrossRefGoogle Scholar
  33. Pinto-Alphandary H., Andremont A., Couvreur P. 2000. Targeted delivery of antibiotics using liposomes and nanoparticles: research and applications. Int. J. Antimicrob. Agents 13:155–168PubMedCrossRefGoogle Scholar
  34. Rafati S., Baba A.A., Bakhshayesh M., Vafa M. 2000. Vaccination of BALB/c mice with Leishmania major amastigote-specific cysteine proteinase. Clin. Exp. Immunol. 120:134–138PubMedCrossRefGoogle Scholar
  35. Read S.M., Northcote D.H. 1981. Minimization of variation in the response to different proteins of the Coomassie blue G dye-binding assay for protein. Anal. Biochem. 116:53–64PubMedCrossRefGoogle Scholar
  36. Rigaud J.L. 2002. Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals. Braz. J. Med. Biol. Res. 35:753–766PubMedCrossRefGoogle Scholar
  37. Rigaud J.L., Lévy D., Mosser G., Lambert O. 1998. Detergent removal by non-polar polystyrene beads. Eur. Biophys. J. 27:305–319CrossRefGoogle Scholar
  38. Sacks D., Noben-Trauth N. 2002. The immunology of susceptibility and resistance to Leishmania major in mice. Nat. Rev. Immunol. 2:845–858PubMedCrossRefGoogle Scholar
  39. Santos H.L., Ciancaglini P. 2000. A practical approach to the choice of a suitable detergent and optimal conditions for solubilizing a membrane protein. Biochem. Educ. 28:178–182CrossRefGoogle Scholar
  40. Seddon A.M., Curnow P., Booth P.J. 2004. Membrane proteins, lipids and detergents: not just a soap opera. Biochim. Biophys. Acta 1666:105–117PubMedCrossRefGoogle Scholar
  41. Shakushiro K., Yamasaki Y., Nishikawa M., Takakura Y. 2004. Efficient scavenger receptor-mediated uptake and cross-presentation of negatively charged soluble antigens by dendritic cells. Immunology 112:211–218PubMedCrossRefGoogle Scholar
  42. Silvius J.R. 1992. Solubilization and functional reconstitution of biomembrane components. Annu. Rev. Biophys. Biomol. Struct. 21:323–348PubMedCrossRefGoogle Scholar
  43. Soong L., Duboise S.M., Kima P., McMahon-Pratt D. 1995. Leishmania pifanoi amastigote antigens protect mice against cutaneous leishmaniasis. Infect. Immun. 63:3559–3566PubMedGoogle Scholar
  44. Tempone A.G., Perez D., Rath S., Vilarinho A.L., Mortara R.A, de Andrade H.F. Jr. 2004. Targeting Leishmania (L.) chagasi amastigotes through macrophage scavenger receptors: the use of drugs entrapped in liposomes containing phosphatidylserine. J. Antimicrob. Chemother. 54:60–68PubMedCrossRefGoogle Scholar
  45. Uemura A., Watarai S., Ohnishi Y., Kodama H. 2005. Protective effect of antiganglioside antibodies against experimental Trypanosoma brucei infection in mice. J. Parasitol. 91:73–78PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Fabiana R. Santos
    • 1
  • Denise B. Ferraz
    • 1
  • Katia R. P. Daghastanli
    • 2
  • F. Juarez Ramalho-Pinto
    • 1
  • Pietro Ciancaglini
    • 2
  1. 1.Departamento de Bioquímica e ImunologiaFaculdade de Medicina de Ribeirão Preto, Universidade de São PauloRibeirão PretoBrasil
  2. 2.Departamento de QuímicaFaculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São PauloRibeirão PretoBrasil

Personalised recommendations